1. Field of the Invention
This invention concerns vaccines, antibodies, proteins, glycoproteins, DNAs and RNAs for prophylaxis and treatment of Cryptosporidium or Cryptosporidium infections. In particular, this invention concerns a Cryptosporidium antigen comprised of a protein with or without carbohydrates attached thereto, as well as polyclonal and monoclonal antibodies directed against the antigen. Additionally, the invention concerns DNA and RNA encoding the Cryptosporidium antigen, mutants, variants and fragments thereof.
2. Background and Related Disclosures
The genus Cryptosporidium consists of Apicomplexan parasites that invade and develop within epithelial cells of the gastrointestinal, hepatobiliary and respiratory tracts of a wide variety of vertebrates including reptiles, birds and mammals. Cryptosporidium was recognized as a cause of animal disease for several decades before the first cases of human cryptosporidiosis were reported in 1976. However, it was not until 1982 that the magnitude of the disease caused by this parasite in both AIDS patients and immunocompetent hosts began to be appreciated. Subsequently, Cryptosporidium has been found to be among the top four causes of human diarrhea worldwide, and to be an increasingly recognized cause of diarrhea in children, animal care workers, and travelers. (Cryptosporidium in Humans, Ed. J. P. Dubai et al., CRC Press, Boca Raton (1990)).
Large waterborne outbreaks of cryptosporidiosis caused by contaminated municipal water supplies in the U.S. and in the UK have been noted in the last ten years (N. Engl. J. Med., 320:1372 (1989), and 33:161 (1994)). The most recent outbreak in Milwaukee in April 1993 involved 400,000 persons and led to the subsequent deaths of more than 100 immunocompromised persons. Like a number of other waterborne outbreaks, the Milwaukee outbreak appears to have been due to contamination from farm or abattoir run-off and specifically due to cryptosporidiosis among cows/calves. Nosocomial transmission in hospitals from patients to staff, patient to patient, and contaminated ice to patients and staff have also been well documented (J. Inf. Dis., 158:647 (1985)).
Waterborne and nosocomial spread reveal a number of biological characteristics of oocysts. First, the infectious dose of the parasite is very low. The ID.sub.50 for human volunteers with normal immune systems is 132 oocysts (New Engl. J. Med., 332:855 (1995)). Second, infected hosts, for example calves, excrete large numbers of oocysts; on the order of 10.sup.10 /day. Third, the oocysts are fully sporulated and ready to infect when excreted. Fourth, the oocysts are environmentally hardy; they remain infectious in cool, moist areas for 3-4 months, and they are not killed by chlorine levels permissible in drinking water. Fifth, the oocysts are quite small, 4-6 .mu.m, and are thus difficult to filter.
The clinical importance of cryptosporidiosis has increased markedly with the recognition of a life-threatening form of the disease in patients with immunodeficiency disorders such as AIDS, hypogammaglobulinemia, and chemotherapeutic immunosuppression. The prevalence of cryptosporidiosis in AIDS patients in the U.S. is estimated to be 5-10% and in central Africa 40%. Immunodeficient patients may have fulminant cryptosporidial diarrhea that may persist until death, whereas the diarrhea of immunocompetent patients is self-limited and rarely lasts more than 2-4 weeks. Cholera-like diarrhea is common in immunocompromised patients with reported losses of up to 17 liters of water per day. Hepatobiliary disease may result in severe abdominal pain and nausea. Removal of immunosuppression in chemotherapy patients leads to resolution of the diarrhea. Occasionally, AIDS patients with cryptosporidiosis will be able to eliminate the parasite after initiation of anti-retroviral therapy (Am. Intern. Med., 116:840 (1992)).
Among those who develop the disease, a quarter have CD4 counts greater than 209, suggesting that the cryptosporidiosis disease may appear relatively early in the course of HIV disease. Unfortunately, few details about the biology of the organisms and the molecular mediators of the disease process have been described and no effective therapy has been discovered.
The infective forms of Cryptosporidium, called sporozoites and merozoites, adhere to the host cell and release the contents of anterior organelles (rhoptries, micronemes or dense granules) during the invasion process (Parasitol. Today, 8:28(1992)). Proteins involved in these events have in many instances been found to be the target of invasion blocking immunity in vitro and neutralization in vivo (ibid). Active and passive immunization studies using malaria and Toxoplasma challenged or infected hosts, have shown that certain secreted components of the apical complex organelles are the target of protective antibodies in these related Apicomplexan parasites. In some cases, as for example in the case of the circumsporozoite and merozoite surface proteins of malaria, these antigens are under development as vaccines.
While the actual interaction between Cryptosporidium and the host's immune system is poorly understood, it is known that disruption of either the cellular or the humoral components can result in protracted cryptosporidiosis (Parasitol. Today, 8:24 (1992)). However, specific antibodies alone neutralize the organism's infectivity. In vitro and in vivo observations indicate that antibodies to Cryptosporidium parvum inhibit invasion and intracellular development leading to protection in challenge experiments, or amelioration of infection in established disease (Infect. Immun., 59:1172 (1991)).
One source of such antibodies is hyperimmune bovine colostrum (HBC) collected from cows immunized with Cryptosporidium oocysts. Calves challenged with Cryptosporidium oocysts are protected from the development of the disease by the administration of HBC (Infect. Immun., 61:4079 (1993)). Some immunocompromised AIDS patients infected with Cryptosporidium have also responded to HBC with a reduction in, or disappearance of, the symptoms of the disease (Gastroenterology, 98:486 (1990)). Immunoglobulin from HBC (HBC Ig) has been found to inhibit the ability of the sporozoite to invade and/or develop intracellularly in vitro and it has been used to immunoprecipitate at least 22 different surface radioiodinated proteins of Cryptosporidium sporozoites. Western blot analysis of proteins of whole oocysts, which contain sporozoites, indicates that HBC predominantly recognizes two proteins of sizes 250 kDa and &gt;900 kDa (Infect. Immun., 61:4079 (1993)).
The use of HBC for human use is problematic. HBC produced using whole oocysts is batch dependent and this may lead to the development of passive immune preparations which are not uniform in immunogenicity and potency. This generates a problem when these immune preparations are to be administered to human patients as such non-uniformity may result in failure of protection. In addition, it would be desirable to allow preparation of large amounts of antigen expressed in heterologous systems rather than to purify the oocyst.
Thus, there is a continuous need for immunogenic agents which are reasonably reproducible and have uniform and controllable immunogenicity and potency, which agents would be useful for the active and passive immunotherapy of cryptosporidiosis in both uncompromised and immunocompromised subjects, such as AIDS patients.
Additionally, there is a need to have available methods for reproducible expression of specific targets for Cryptosporidium antigens in large amounts, which antigens would provide a better immunogen. This approach requires that specific Cryptosporidium antigen genes are cloned and identified as potential candidates through their ability to elicit an antibody response that is immunoprotective. Before antibodies produced in this manner are tested in or administered to humans or animals, testing in in vitro assay of its inhibitory effect on invasion or intracellular development of the Cryptosporidium organism in cultured cells, and in vivo studies, would be desirable.
It is, therefore, a primary objective of this invention to provide polyclonal or monoclonal antibodies to be used for prophylaxis and treatment of cryptosporidiosis and to express a portion of the GP900 sequence/locus to provide target protein antigens allowing production of recombinant anti-Cryptosporidium vaccines and passive immune products.
All patents, patent applications and publications cited herein are hereby incorporated by reference.